Evidence of extensive phospholipid fatty acid methylation during the assumed selective methylation of plasma free fatty acids by diazomethane

We compared a conventional method (Method I) for measuring plasma free fatty acid (FFA) concentrations with two more rapid procedures (Method II and Method III). Method I required total lipid extraction, separation of FFA by thin-layer chromatography, methylation, and gas-liquid chromatographic analysis of the fatty acid (FA) methyl esters. Method II was a colorimetric procedure. Method III relied upon diazomethane's presumed ability to selectively methylate FFA even in the presence of FA esters. The three methods were compared using plasma from fasted and from fed nude mice, tumor-bearing mice (MX-1 and ZR-75-1 human mammary carcinomas), and controls. Method II, was less reliable than Method I, but both gave similar mean values for plasma FFA levels in fasted mice. Both Methods I and II also showed similar lowering of plasma FFA levels after feeding previously fasted mice. Method III consistently gave values that were far greater than those obtained using Methods I and II. Moreover, highly significant differences between fasted and fed mice were obscured by direct methylation of plasma FFA with diazomethane (Method III). The excess FA methyl esters formed in Method III were derived from plasma phospholipids, but not from plasma triacylglycerols. After feeding fasted mice, plasma free palmitic acid and oleic acid levels fell (Method I); by contrast, the excess “FFA” formed by methylation of plasma phospholipid FA increased two-fold and fourteen-fold, respectively. Caution is therefore advised in the use of direct methylating agents when measuring total and individual plasma FFA levels.     

Antioxidant-restricted diet reduces plasma nonesterified fatty acids in trained athletes

Nonesterified FA (NEFA) are a major fuel source for humans at rest and during moderate exercise. The effect of dietary antioxidant restriction on plasma NEFA levels and exercise performance in trained athletes was examined. Seventeen athletes followed a 2-wk restricted-antioxidant (R-AO) diet, which resulted in a threefold reduction in antioxidant intake (ascorbic acid, 139 to 49 mg; β-carotene, 5093 to 1142 μg) and a significant (P=0.001) reduction in the plasma NEFA. The amount and types of fat consumed were not different between the R-AO and habitual diets. Exercise time to exhaustion was not affected by the R-AO diet, but rating of perceived exertion (RPE) was significantly (P=0.03) elevated. The increase in RPE may have occurred as a result of the R-AO diet and subsequent reduction in plasma NEFA; however, further research is required to confirm this conclusion.     

Mono- and polyenoic acid distribution in plasma nonesterified fatty acids in Kwashiorkor

Plasma NEFA levels were determined in Kwashiorkor (17 cases) and normal infants (13 cases) by Dole’s method. The distribution of mono- and polyenoic acids was investigated by Abdel-Wahab’s method. Plasma NEFA levels were found to be significantly lower in Kwashiorkor infants (445 μEq/L±32) than in normals (591 μEq/L±66). Plasma oleic, linoleic and linolenic acids were detected in measurable amounts in both Kwashiorkor and normals, but were significantly higher in Kwashiorkor. Arachidonic acid was hardly detectable in either group. The high levels of mono- and polyenoic acids in Kwashiorkor can be attributed to demands on them for the synthesis of fat and/or preferential utilization of the unsaturated fatty acids in mobilization of other lipid components.     

Elevated levels of nonesterified fatty acids in the myocardium of alloxan diabetic rats

Myocardial nonesterified fatty acids (NEFA) increase markedly within the first two days after the induction of insulin-dependent diabetes mellitus in rats by intravenous injection of alloxan. After initial variability, NEFA levels in diabetic hearets remain constant at approximately 450 nmol/g tissue (16 nmol/gmmol lipid P), which is about three times higher than that in control hearts. Nonesterified linoleic acid is significantly increased in diabetic heart whereas both arachidonic and docosahexaenoic acids are decreased compared to controls.     

The effects of tricaine methanesulfonate (MS-222) on plasma nonesterified fatty acids in rainbow trout,Oncorhynchus mykiss

The effects of tricaine methanesulfonate (MS-222), a commonly used fish anesthetic, on plasma nonesterified fatty acid levels (NEFA) were examined in rainbow trout,Oncorhynchus mykiss. Total NEFA levels declined with increasing duration of exposure to MS-222. Most of the decline in total NEFA was due to decreases in saturated fatty acids (14∶0, 16∶0 and 18∶0). The fatty acid displaying the most rapid response to exposure to MS-222 was 20∶5n−3. The lower plasma NEFA levels in anesthetized fish may be explained by depressed lipolysis in the presence of the anesthetic.     

A semiautomated enzymatic method for determination of nonesterified fatty acid concentration in milk and plasma

An enzymatic assay for the determination of non-esterified fatty acid concentrations in milk and plasma is described. The procedure is semiautomated for use with a plate luminometer or plate spectrophotometer and enables routine batch processing of large numbers of small samples (≤5 μL). Following the activation of nonesterified fatty acids (NEFA) by acyl-CoA synthetase, the current assay utilizes UDP-glucose pyrophosphorylase to link inorganic pyrophosphate to the production of NADH through the reactions catalyzed by phosphoglucomutase and glucose-6-phosphate 1-dehydrogenase. With this assay sequence the formation of NADH from NEFA is complete within 50 min at 37°C. Enzymatic spectrophotometric techniques were unsuitable for NEFA determination in human milk due to the opacity of the sample. The use of the NADH-luciferase system has overcome this problem, allowing the enzymatic determination of NEFA in human milk. Sample collection and treatment procedures for milk and plasma have been developed to prevent enzymatic lipolysis and to limit interference from enzymes present in milk. The recovery of palmitic acid added to milk and plasma samples was 94.9±2.9 and 100±4.5%, respectively. There was no difference (P=0.13) in plasma NEFA concentrations determined by the current method and a commercially available enzymatic spectrophotometric technique (Wako NEFA-C kit). Plasma NEFA concentrations determined by gas chromatography were 28% higher compared to both the Wako NEFA-C kit and the current method.     

Digestion and absorption of free and esterified fish oil fatty acids in rats

This study was designed to test the hypotheses that digestibility and post-absorption metabolism of fish oil are influenced by impaired lipolysis and by the stereospecific composition of its triacylglycerols. Male Wistar rats were fed nonpurified diets containing one of the following fat sources: 9% native fish oil (NFO), 9% autorandomized fish oil (RFO), 8.1% fish oil-derived free fatty acids (FO-FFA) plus 0.9% glycerol, or 9% soybean oil (SO) as a reference fat. In a 24-day balance study, apparent digestibility of total dietary fat averaged 93.1% in the SO, NFO and RFO groups, and 90.9% in the FO-FFA group. Randomization of fish oil had no effect on apparent digestibility of individual fatty acids. In rats fed FO-FFA, apparent absorption of saturated and monounsaturated fatty acids was lower when compared to the NFO and RFO groups. Feeding the FO-FFA diet tended to increase plasma triglyceride content. The hypocholesterolemic effect of polyunsaturated n−3 fatty acids was not influenced by the dietary source. Similar effects on fatty acid profiles of plasma and liver phospholipids were caused by the NFO, RFO and the FO-FFA diets. We conclude that once polyunsaturated n−3 fatty acids are absorbed, their effect on lipid metabolism is not determined by the dietary source.     

Adsorption equilibria of fatty acids between methanol/water and reversed-phase chromatographic adsorbents

The adsorption equilibria are discussed for fatty acids 16∶1n?7, 16∶2n?4, and 20∶5n?3 (eicosapentaenoic acid, EPA). These fatty acids are major components of a polyunsaturated fatty acid concentrate from the microalga Phaeodactylum tricornutum. The solvents used were methanol/water (1% acetic acid) mixtures of different compositions, and the adsorbents used were chromatographic reversed-phases octylsilyl C₈, octadecylsilyl C₁₈, and dodecylsilyl C₂₂ of different particle and pore sizes. The kinetic studies showed that equilibrium was attained instantaneously, suggesting an absence of mass transfer limitations. The equilibrium data were fitted by the Freundlich isotherm. The separation efficiency of EPA from 16∶1n?7 and 16∶2n?4 in all the adsorbent-solvent systems was compared in terms of the separation factors α_EPA/16:2n-4=K _EPA/K _16:2n-4 and α_16:1n-7/EPA=K _16:1n-7/K _EPA, where K _i is the fatty acid distribution ratio between the stationary and the liquid phases. The EPA separation from 16∶1n?7 and 16∶2n?4 by liquid chromatography could be predicted using the Craig model for the various solvent-adsorbent combinations. The best adsorbents for purifying EPA were: C₁₈, PEP, 8 μm, 100 Å, and C₂₂, 10 μm, 100 Å, and the best solvent was methanol/water (1% acetic acid) 75∶25, w/w.     

Specific modifications of phosphatidylinositol and nonesterified fatty acid fractions in cultured porcine cardiomyocytes supplemented with n-3 polyunsaturated fatty acids

Mechanisms for the antiarrhythmic effect of n−3 polyunsaturated fatty acids (PUFA) are currently being investigated using isolated cardiac myocytes. It is still not known whether the incorporation of n−3 PUFA into membrane phospholipids is a prerequisite for its protective action or if n−3 PUFA exert antiarrhythmic effects in their nonesterified form as demonstrated by recent studies. Adult porcine cardiomyocytes were grown in media supplemented with arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). After 24 h, analysis of total lipids showed that the myocytes were enriched with the respective fatty acids compared to control cells. Large proportions of all three fatty acids supplemented (69% AA, 72% DHA, and 66% EPA) remained unesterified. Fatty acid analysis of total phospholipids (PL) revealed that the incorporation of EPA and DHA, though small, was significantly different (P<0.05) from that of the control cells. The PL fraction was further separated into phosphatidylinositol (Pl), phosphatidylethanolamine, phosphatidylcholine, and phosphatidylserine to study the pattern of incorporation of the fatty acids in these fractions. It became apparent that EPA and DHA were selectively incorporated into the Pl fraction. This study demonstrates that in adult porcine cardiomyocytes, the n−3 PUFA supplementation selectively modulates two important lipid fractions, nonesterified fatty acid and Pl, which were implicated in the mechanisms of prevention of cardiac arrhythmias.     

HZSM-5分子筛上甲苯择形甲基化反应积炭行为探讨

将水热合成法制备并改性的HZSM-5分子筛催化剂应用于甲苯甲醇甲基化反应,催化剂表现出良好的甲苯转化率和二甲苯对位选择性。采用程序升温氧化法、固体紫外-可见吸收光谱、核磁共振法和气相色谱串联质谱法等研究催化剂的积炭行为。结果表明,反应介质甲醇中的氧未参与积炭形成,积炭成分较为单一,均由碳氢两种元素组成。积炭分为可溶的芳烃和稠环芳烃及其衍生物以及不可溶稠环芳烃、石墨前驱体、石墨等中的一种或几种混合物。甲苯与甲醇作为反应底物同时存在也决定了可溶性成分中单甲基或多甲基取代芳烃占大多数。早期积炭主要分布于分子筛孔道,积炭组成和分布由分子筛孔道结构和外表面酸性、反应物性质与反应条件决定。     

Rapid analysis of fatty acids in plasma lipids

A rapid and convenient procedure for the quantitative determination of the fatty acid composition of plasma lipids is described. Human plasma was applied directly to the preadsorbent zones of thin-layer silica gel plates with added antioxidant, internal standards and carriers. The thin-layer chromatography (TLC) plates were partially developed with methanol followed by chloroform/methanol (1∶1, v/v), and then they were fully developed in hexane/diethyl ether/acetic acid (80∶20∶1, v/v/v) to separate the major classes of lipids. Silica gel from regions containing the separated lipids was scraped into screw-capped tubes and treated with boron trifluoride-methanol prior to gas chromatography. The method of direct application to TLC plates gave yields and compositions of fatty acids very similar to the method of applying extracted plasma lipids. This relatively simple method is suitable for analyzing the fatty acids in plasma lipids from a 50 microliter finger-tip blood samples from an individual, and it may be useful in wide-scale screening of different individuals to estimate the relative amounts of ingested polyunsaturated fatty acids. Pfizer Biomedical Research Awardee.     

Use of dried blood for measurement of trans fatty acids

Background  

Fatty acid measurements especially trans fatty acid has gained interest in recent times. Among the various available biomarkers, adipose tissue is considered to be the best for the long term dietary intake but the invasive nature of tissue aspiration reduces its utility. Phlebotomy is a much less invasive method of sample collection when a large number of participants are involved in the study and therefore is an alternative, most suitable for large population based studies. In the present study fatty acid (with special emphasis on trans fatty acid) extraction from blood spotted and dried on filter paper was carried out to simplify the sample collection procedure and transportation.     

Cellulose esterification with fatty acids and acetic anhydride in lithium chloride/N,N-dimethylacetamide medium

Homogeneous esterification of cellulose with saturated fatty acids (n-octanoic to n-octadecanoic) was accomplished with acetic anhydride co-reactant in lithium chloride/N,N-dimethylacetamide (LiCl/DMAc) medium. Cellulose mixed triesters (CMT) were obtained after 5 h at 130°C with an average of 2.2 acetyl groups and 0.8 fatty substituents per anhydroglucose unit. A mixed acetic-fatty anhydride, formed in situ, accounts for the grafting of the fatty moiety. The purified products were characterized and compared to the analogous cellulose simple fatty triesters (CST) that were synthesized from fatty acid chlorides in pyridine medium. Dynamic contact angle with water, glass transition, and storage moduli were correlated with the length of the fatty substituents. The CMT proved to be highly hydrophobic and more mechanically resistant than the CST.     

甲醇-脂肪酸混合物临界参数及酯交换反应条件探讨

超临界甲醇法制备生物柴油反应涉及到拟二元混合物临界参数的计算。操作参数对系统热力学相态的影响直接关系到化学反应的机理,进而决定反应速度和产物转化率。为此,采用基团贡献法与L-B混合规则对甲醇和脂肪酸混合物的临界参数进行了计算。发现采用C-G基团贡献法与L-B混合规则可以较好地描述甲醇和大豆油混合物的临界参数,并且无需考虑相互作用参数。与文献值相比,平均误差为1.7%。据此,对反应器内混合物相态进行分析,讨论了低醇油摩尔比、反应温度和反应压力等条件对反应物相态和甲酯产率的影响。结果对于指导高压法制备生物柴油工艺设计有所帮助。     

Long-chain polyunsaturated fatty acids in plasma lipids of obese children

Fatty acid composition of plasma phospholipids (PL), triglycerides (TG), and sterol esters (STE) was determined by high-resolution capillary gas-liquid chromatography in 22 obese children (age: 13.7±1.4 y, body weight relative to normal weight for height: 170±24%, mean ±SD) and compared with data obtained in 25 age-matched healthy controls. There were no differences in the levels of linoleic acid (LA, C_18∶2n-6) in any of the plasma fractions from the obese children and the controls. Obese children exhibited significantly higher values of arachidonic acid (AA, C_20∶4n-6) than controls both in PL (12.6 [2.4] vs. 8.3 [1.4], % wt/wt, [median (interquartile range)],P<0.001) and STE (7.3 [1.8] vs. 6.0 [1.1],P<0.05). Similarly, obese children showed higher values than controls for dihomo-γ-linolenic acid (DHGLA, C_20∶3n-6) in PL (4.0 [0.5] vs. 3.0 [0.6],P<0.001), TG (0.4 [0.1] vs. 0.2 [0.1],P<0.001), and STE (0.9 [0.1] vs. 0.7 [0.1],P<0.01), and for γ-linolenic acid (C_18∶3n-6) in STE (1.1 [0.2] vs. 0.8 [0.2],P<0.001). The AA/LA ratios were higher in obese children than in controls in PL (0.68 [0.16] vs. 0.42 [0.09],P<0.0005) and STE (0.16 [0.04] vs. 0.12 [0.02],P<0.05), whereas the AA/DHGLA ratios were lower in TG of obese children than in controls (3.40 [0.64] vs. 5.10 [1.75],P<0.005). Plasma glucose concentrations were inversely related to AA in TG (r=0.53,P<0.05), and plasma TG concentrations were inversely related to AA in PL and STE (r=−0.49,P<0.05 andr=−0.48,P<0.05) and to the AA/DHGLA ratios in PL (r=−0.57,P<0.01),TG (r=−0.56,P<0.01) and STE (r=−0.56,P<0.01). We conclude that the significantly higher values of n-6 long-chain polyunsaturated fatty acids (LCP) in plasma lipids of obese children than in age-matched controls may be caused by an enhanced activity of Δ6-desaturation, and we speculate that elevated fasting immunoreactive insulin seen in obese children (19.4±8.0 μU/mL) may stimulate synthesis of n-6 LCP fatty acids.     

Accelerated solvent extraction for quantitative measurement of fatty acids in plasma and erythrocytes

Consumption of fish rich in n-3 highly unsaturated FAs (i.e., EPA and DHA) has been suggested to decrease the risk of lifestyle-related diseases such as coronary heart disease, cancer, diabetes, and dementia. Blood eevels of those FA are known appropriate biomarkers of both the corresponding dietary FA intakes and fish consumption. In place of traditional handwork methods for extracting FA, we performed an accelerated solvent extraction (ASE) for at least 13 selected FA in plasma and erythrocytes to measure them by GLC. The FA levels (concentrations and compositions) in 35–50 μL of plasma or erythrocytes were extracted by ASE and measured by GLC. Intra-and interassay coefficients of variation were≤6.0% for both blood materials, except with a minor group of FA (≤1.0% of total FA). When ASE was compared with two traditional handwork methods, FA levels in plasma from 18 healthy subjects were all coincident with very high Pearson's correlation coefficients for the three sets of the sama 18 samples (r≤0.85 to 0.95, P<0.0001), except for 18∶0 (r=0.59, P<0.01). Using ASE and GLC, we have developed a new method for determination the levels of FA in plasma and erythrocytes as biomarkers for dietary intake of fish, fat, and FA. This new method makes it feasible to measure small volumes of samples, automatically, quantitatively, routinely, easily, rapidly and cheaply, with acceptable precision and accuracy.     

Intestinal metabolism of plasma free fatty acids in streptozotocin diabetic rats

Moderate insulin deficiency was reported to be accompanied by an increased production of intestinal very low density lipoprotein (VLDL) triglyceride in the rat. Because plasma free fatty acids (FFA) are incorporated into triglyceride by intestinal mucosa of rats and humans and plasma FFA are increased in insulin-deficient diabetes mellitus, we investigated several aspects of the intestinal metabolism of plasma FFA in diabetic rats. All experiments were performed on the third day following the i.v. injection of streptozotocin (45 mg/kg body weight) or buffer alone. A (¹⁴C)palmitic acid-rat serum complex was rapidly injected intravenously and its initial uptake by small bowel mucosa, the intracellular incorporation into lipids and water soluble metabolites and the specific radioactivity of triglycerides of mucosal homogenates was determined. No significant differences could be found between diabetic and control rats at 2 and 5 min after¹⁴C-palmitate i.v., suggesting that neither the influx of plasma free fatty acids into intestinal mucosal cells nor their initial intracellular metabolic pathways are significantly altered in moderately diabetic rats. A pronounced decrease in intestinal mucosal triglyceride at 10 min after¹⁴C-palmitate i.v. might be interpreted as indirect evidence for an enhanced triglyceride efflux from intestinal mucosa into mesenteric lymph in diabetic rats.     

Use of aqueous HCI/MeOH as esterification reagent for analysis of fatty acids derived from soybean lipids

A quick, reliable and very inexpensive method is described for the analysis of fatty acids derived from soybean lipids. The method involves extraction of soybean lipids with petroleum ether, followed by hydrolysis of lipids with KOH/MeOH (0.5 M) for 5 min at 100 C followed by esterification with aq. HC1 (36%)/MeOH (4:1, v/v) for 15 min at 100 C. No problems were encountered with the esterifica-tion procedure in the presence of water and the procedure gave results comparable to die more conventional BF₃/MeOH reagent. The aq. HCI/MeOH reagent is several hundred times cheaper than BF₃/MeOH, and does not compromise the efficiency of the reagent.